the authors disclose no potential conflicts of...
TRANSCRIPT
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KEAP1 Is a Redox Sensitive Target That Arbitrates the Opposing
Radiosensitive Effects of Parthenolide in Normal and Cancer Cells
Yong Xu1, Fang Fang2, Sumitra Miriyala1, Peter A. Crooks3, Terry D. Oberley4, Luksana
Chaiswing4, Teresa Noel1, Aaron K. Holley1, Yanming Zhao1, Kelley K. Kiningham5, Daret K.
St. Clair1† and William H. St. Clair2†
1Graduate Center for Toxicology, 2Department of Radiation Medicine, University of Kentucky,
Lexington, KY 40536; 3Department of Pharmaceutical Sciences, UAMS College of Pharmacy,
Little Rock, AR 72205; 4Department of Pathology, University of Wisconsin, Madison, WI
53705; and 5Department of Pharmaceutical Sciences, Belmont University School of Pharmacy,
Nashville, TN 37212
Running title: Keap1, a central regulator of cellular redox signaling
Key words: Keap1, Nrf2, PGAM5, Bcl-xL, parthenolide, radiotherapy, prostate cancer, reactive
oxygen species (ROS), redox modification, antioxidant proteins, mitochondrial function
† Corresponding Authors: William H. St. Clair, M.D., Ph.D., Department of Radiation Medicine,
University of Kentucky, College of Medicine, Lexington, KY 40536; Phone: 1-(859) 257-4931;
Fax: 1-(859) 323-6486 ; Email: [email protected]. Daret K. St. Clair, Ph.D., Graduate Center for
Toxicology, University of Kentucky, College of Medicine, Lexington, KY 40536; Phone: 1-
(859) 257-3720; Fax: 1-(859) 323-1059; Email: [email protected].
The authors disclose no potential conflicts of interest
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Abstract
Elevated oxidative stress is observed more frequently in cancer cells than in normal cells. It
is therefore expected that additional exposure to a low level of reactive oxygen species
(ROS) will push cancer cells toward death, whereas normal cells might maintain redox
homeostasis through adaptive antioxidant responses. We previously demonstrated that
parthenolide enhances ROS production in prostate cancer cells through activation of
NADPH oxidase. The present study identifies KEAP1 as the downstream redox target that
contributes to parthenolide's radiosensitization effect in prostate cancer cells. In vivo,
parthenolide increases radiosensitivity of mouse xenograft tumors but protects normal
prostate and bladder tissues against radiation-induced injury. Mechanistically,
parthenolide increases the level of cellular ROS and causes oxidation of thioredoxin (TrX)
in prostate cancer cells, leading to a TrX-dependent increase in a reduced state of KEAP1,
which in turn leads to KEAP1-mediated PGAM5 and Bcl-xL (BCL2L1) degradation. In
contrast, parthenolide increases oxidation of KEAP1 in normal prostate epithelial cells,
leading to increased Nrf2 (NFE2L2) levels and subsequent Nrf2-dependent expression of
antioxidant enzymes. These results reveal a novel redox-mediated modification of KEAP1
in controlling the differential effect of parthenolide on tumor and normal cell
radiosensitivity. Further, they show it is possible to develop a tumor-specific
radiosensitizing agent with radioprotective properties in normal cells.
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Introduction
It is well-documented that cancer cells are usually under more oxidative stress than normal cells
are, in part due to a hyperactive metabolism that fuels their rapid growth (1, 2). Thus, a therapy
designed to increase reactive oxygen species (ROS) to a level above the threshold for cancer cell
death, but to an adaptable level for normal cells, would be an attractive strategy to selectively kill
cancer cells (3, 4). Redox homeostasis is thought to regulate many cellular processes that are
essential for maintenance of normal physiological conditions but is aberrantly modulated in
cancers (5, 6). The functions of ROS are both beneficial and deleterious due to their dual role in
the prosurvival and antisurvival pathways. As a secondary messenger in cell signaling, ROS are
required for normal development and can initiate adaptive responses in cellular defense (7, 8).
On the other hand, ROS cause structural damage and functional decline in DNA, proteins and
lipids, and consequently act as an anti-tumorigenic factor by inducing cell senescence and
apoptosis (9, 10). Indeed, ROS-mediated cell death is an important basis for radiotherapy and
many chemotherapeutic treatments (11, 12). Currently, these therapeutic strategies are being
used to kill cancer cells without benefit of a rational design that exploits the intrinsic differences
in the cellular redox status of normal cells and cancer cells.
Antioxidant defense systems are essential for the regulation of ROS levels, which have an
important function in the maintenance of cellular redox hemostasis. Mounting evidence
demonstrates that a decline in antioxidant function may be involved in tumorigenesis due to
prooxidant conditions that result from ROS accumulation. For example, manganese superoxide
dismutase (MnSOD) is down-regulated in many types of cancer, and overexpression of MnSOD
results in suppression of tumorigenesis (13-15). High levels of antioxidants caused by therapy-
mediated activation of pro-survival pathways, such as NF-κB and Nrf2, are thought to protect
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cancer cells against treatment (16-18). Thus, inhibition of prosurvival pathways has been a
traditional strategy to enhance therapeutic efficacy.
Increasing evidence demonstrates that certain mild prooxidant compounds derived from
natural herbal medicines might enhance some anticancer treatments by modulating the redox
state of cancer cells to high prooxidant levels (19, 20). Parthenolide, an active ingredient derived
from the traditional anti-inflammatory medical plant feverfew (Tanacetum parthenium), belongs
to the family of sesquiterpene lactones containing an α-methylene-γ-lactone moiety and an
epoxide group, which is able to conjugate thiols of proteins through a Michael addition reaction
(21). In addition to its anti-inflammatory effect, parthenolide appears to be toxic to a variety of
cancer cells (22-25). Importantly, parthenolide has no cytotoxic effect on normal cells (25).
Mechanistically, parthenolide has been shown to increase apoptosis in cancer cells through
inhibition of multiple prosurvival pathways, such as NF-κB and PI3K-AKT (19, 26). However,
these findings do not explain why parthenolide is not toxic to normal cells.
Post-translational modification is a key mechanism by which proteins dramatically
increase their functional diversity. Reversible redox modification of protein cysteine residues
plays an important role in vital cell signaling pathways related to many physiological and
pathogenic processes (27, 28). The Keap1-Nrf2 pathway is one of the main cellular defense
mechanisms against oxidative stress (29, 30). The present study examines the role of cysteine
modifications in modulating radiation responses in prostate cancer cells versus normal prostate
epithelial cells. It elucidates the functional link between redox modulation and cell signaling
transduction pathways, and it provides evidence for the differential effect of parthenolide on
cellular redox status in normal and cancer cells. The results reveal that parthenolide-mediated
redox modification of Keap1 serves as a central regulator of differential responses to radiation
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therapy in normal and tumor cells. The present study provides a proof-of-concept for utilizing an
intrinsic difference in cellular redox conditions to kill tumor cells while protecting normal cells
from the unwanted side effects of radiation.
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Materials and Methods Cell culture, cell transfection, treatment, and cell survival analysis. Human prostate
carcinoma/adenocarcinoma LNCaP, PC3 and DU145 cell lines as well as human prostate
epithelial viral transformed PZ-HPV-7 (PZ) and RWPE-1 cell lines were obtained from
American Type Culture Collection (ATCC). Normal prostate epithelial PrEC cells were
purchased from Lonza. All cell lines were cultured and maintained in the media recommended in
the manufacturer’s protocols. Plasmid cloned Keap1 cDNA and Bcl-xL cDNA (OriGene) and
siRNAs for knocking down Keap1, Nrf2, thioredoxin (TrX), PGAM5, and Bcl-xL (Dharmacon)
were transfected into cultured cells prior to treatment. Parthenolide and its water soluble prodrug,
dimethylamino-parthenolide (DMAPT), were synthesized as previously described (31). The cells
were treated with 0-5 µM parthenolide followed by irradiation by a 250 kV X-ray machine
(Faxitron X-ray Corp.) with peak energy of 130 kV, 0.05 mm Al filter, at a dose of 0 to 6 Gy.
Cell survival rates were quantified by colony survival fraction, Trypan blue exclusion assay, and
MTT assay, as previously described (25, 32).
Animals. Four- to five-week-old male NCRNU (nu/nu athymic nude) mice were obtained from
Taconic (Hudson). For formation of xenograft tumors, 1.8 x 106 cells mixed in Matrigel (BD
Biosciences) were subcutaneously injected into the right flank of the mice. Tumor volumes were
routinely measured and their sizes calculated based on a protocol described elsewhere (33).
Animals with an average tumor size of 500 mm3 were randomized into several groups for
DMAPT and radiation treatments. The tumors were treated five times with 10 mg/kg DMAPT
and 3 Gy IR. The tumor tissues were collected, and 100 µg of tissue were lysed to quantify
amounts of oxidized or reduced Keap1 and levels of downstream proteins. To determine the
protective effect of parthenolide against radiation damage, mice were pretreated with DMAPT
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followed by radiation treatment (5 x 3Gy). Prostate and bladder tissues were fixed, embedded
and processed for routine Electron microscopy (EM). The embedded blocks were sectioned and
transferred to copper grids and counterstained with uranyl acetate, followed by lead citrate. Grids
were observed in an electron microscope (Hitachi H-600) operated at 75 kV. Mitochondrial
damage was quantified by a pathologist (TDO) using identified ultrastructural changes including
swelling, vacuolization, myelination, disorganization and loss of cristae, lysosomal degradation,
and membrane disruption. Animal experimental procedures were approved by the Institutional
Animal Care and Use Committee of the University of Kentucky, Approval Protocol No.
01077M2006.
Quantification of intracellular superoxide and prooxidant. Dihydroethidium (DHE,
Invitrogen), which exhibits blue fluorescence in the cytosol until oxidized, was used to estimate
the levels of superoxide after parthenolide treatment. To confirm the level of superoxide induced
by parthenolide, the cells were pretreated with 50 μg PEG-SOD (Sigma) for 1 h followed by
parthenolide treatment. Antimycin (Sigma) was used as a positive control because it has been
shown to increase superoxide in all tested cell lines. A dichlorofluorescein (DCF) assay was used
to quantify the levels of intracellular ROS after parthenolide treatment. The cells were labeled by
both carboxy-H2DCFDA (sensitive to oxidation, Invitrogen) and carboxy-DCFDA (insensitive to
oxidation, Invitrogen). The H2DCFDA:DCFDA ratio was used to optimize the controls of cell
number, dry uptake, and ester cleavage. The procedures for both DHE and DCF were performed
by the University of Kentucky Flow Cytometer Facility using a FACScan protocol provided by
Dr. Douglas R. Spitz (34).
Measurement of oxygen consumption rates (OCR). To determine how parthenolide changes
mitochondrial function in cancer and normal cells, a Seahorse Bioscience XF24 Extracellular
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Flux Analyzer was used to measure OCR after parthenolide treatment. The XF24 creates a
transient, 7-μl chamber in specialized microplates, which allows determination of oxygen and
proton concentrations in real time. To allow comparison between experiments, data are
expressed as the OCR in pmol/min or the rate of extracellular acidification in mpH/min. Reserve
capacity, an important index that indicates capacity of mitochondrial respiration, is calculated by
subtracting baseline OCR from maximal OCR.
Detection of oxidized and reduced forms of Keap1 protein. 3-N-maleimido-propionyl
biocytin was used to selectively label sulfhydryl (SH) and then was detected by biotin-
streptavidin integration on the blots, as previously described (35). To quantify disulfide (S-S)
bonds, the SH form was stabilized by treating with N-ethylmaleimide and then the S-S bonds
were reduced by treating with 2-mercaptoethanol. To identify SH and S-S moieties of Keap1
protein, the labeled proteins were immunoprecipitated by Keap1 antibody (abcam) and subjected
to SDS-PAGE, followed by detection with horseradish peroxidase-conjugated streptavidin
(Sigma).
Immunoblots and immunoprecipitation. Homogenized cells and tumor tissues were
electrophoresed on an 8% (w/v) SDS-PAGE gel, transferred onto a nitrocellulose membrane, and
subsequently incubated with primary antibodies against Keap1 (abcam), Nrf2 (abcam), MnSOD
(Upstate Biotech), CuZnSOD (eBiosci), Gpx (abcam), catalase (Millipore), TrX (BD Sciences),
PGAM5 (Santa Cruz Biotech.), Bcl-xL (Santa Cruz Biotech.), LC3B (cell signaling), β-actin
(sigma), and pCNA (Santa Cruz Biotech.). All secondary antibodies were obtained from Santa
Cruz Biotech. Immunoblots were visualized using an enhanced chemiluminescence detection
system (Amersham Pharmacia Biotech.). For immunoprecipitation, cell extracts were incubated
overnight with one primary antibody at 4ºC and integrated with a protein A/G agarose (Santa
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Cruz Biotech). Immunocomplexes were precipitated and fractionated on an SDS-PAGE gel.
Interacting proteins were detected by immunoblotting with their primary antibodies.
SOD enzymatic assay. MnSOD activities were measured by the nitroblue tetrazolium (NBT)-
bathocuproin sulfonate (BCS) reduction inhibition method. Sodium cyanide (2 mM) was used to
inhibit CuZnSOD activity (36).
Statistical data analyses. Multiple independent experiments were performed for each set of data
presented. Images in Immunoblots were quantified using Carestream Molecular Imaging
software (Carestream Health Inc.). Statistical significance was analyzed using one-way ANOVA
and Tukey's Multiple Comparison Test, followed by data analysis with Graphpad Prism.
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Results
Parthenolide enhances radiosensitivity of prostate tumors but protects normal tissues from
radiation. Our previous studies demonstrated that parthenolide is able to selectively enhance the
radiosensitivity of prostate cancer cells without injury to normal prostate epithelial cells (24, 25).
To determine whether parthenolide enhances radiotherapy in vivo, human prostate cancer PC3
cells were subcutaneously implanted into the right flank of nude male mice. When tumor size
reached a volume of 500 mm3, animals were randomized into groups according to treatment
consisting of saline or 10 mg/kg DMAPT. One hour after saline or DMAPT was administered,
the tumors were treated with fractionated radiation of 1 Gy or 2 Gy per day for 5 days, followed
by routine measurement of tumor volume. The tumor growth curves are shown in Fig. 1A. Mice
were humanely killed when a tumor reached the maximum size of 2000 mm3. Tumor growth was
clearly delayed in the treatment groups, particularly when the drug and radiation were combined,
compared to growth in the untreated group. The tumor growth rates after treatment were
compared according to the days needed for tumor volume to reach 2000 mm3. DMAPT
significantly enhanced radiotherapeutic efficiency compared to the effects of radiation treatment
alone (Fig. 1B). A separate group of nude male mice that had no cancer cell implantation was
treated with DMAPT and radiation to determine the toxicity of DMAPT to organs that can be
affected by radiation therapy of prostate cancer. Prostates and bladders of the animals were
examined by light and EM. At 60 days after irradiation, no gross pathology was observed (data
not shown). However, ultrastructural damage was clearly observed by EM (Fig. 1C).
Mitochondrial damage was most pronounced and this was morphometrically analyzed. The
number of damaged mitochondria in prostate and bladder was proportional to radiation exposure.
Pretreatment with DMAPT significantly reduced the number of damaged mitochondria in both
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organs compared to the group without DMAPT treatment (Fig. 1C and D). These results indicate
that DMAPT, the water soluble prodrug of parthenolide, is a promising agent for selectively
enhancing the sensitivity of prostate cancer cells to radiation while protecting normal tissues
from damage caused by radiation.
Parthenolide differentially modulates cellular ROS levels in cancer and normal cells. To
determine the effect of parthenolide on redox homeostasis in cancer and normal cells, the levels
of superoxide and total ROS after parthenolide treatment were measured using flow cytometry.
The mean fluorescence intensity of DHE and the H2DCFDA:DCFDA ratio were higher in
prostate cancer PC3 cells than in normal prostate PZ and PrEC cells, indicating higher basal
levels of superoxide and total ROS, respectively (Fig. 2A-B). Following parthenolide treatment,
DHE and DCF levels increased further in PC3 cells but declined slightly in both types of non-
cancerous cells. Combining PEG-SOD with parthenolide treatment restored the basal level of
DHE fluorescence. As positive controls, addition of equivalent concentrations of the ROS
stimulating agents antimycin (Fig. 2A) and PMA (Fig. 2B) caused high levels of ROS in all the
tested cell lines.
The levels of antioxidant proteins were also quantified (Fig. 2C). Parthenolide altered the
protein level of the antioxidant enzymes, in particular mitochondria-localized antioxidant
enzymes. MnSOD and glutathione peroxidase (GpX) were significantly reduced in all three
parthenolide-treated prostate cancer cell lines. Intriguingly, parthenolide had the opposite effect
in the three normal prostate cell lines. However, neither cancer nor normal cells showed any
obvious changes after parthenolide treatment for the major cytosolic superoxide removal protein,
copper and zinc-containing SOD (CuZnSOD). This observation was confirmed by quantification
of the corresponding enzyme activity (Fig. 2D). These results suggest that parthenolide-mediated
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alteration of cellular redox status is mediated, at least in part, by changing the activities of
antioxidant enzymes in mitochondria.
To probe whether altering cellular redox status is associated with a change in
mitochondrial respiration, the OCR in the parthenolide-treated cells was measured using a
Seahorse Bioscience FX OxygenFlux Analyzer. The basal and maximal OCR in normal cells
was higher than in cancer cells (Fig. 2E). Importantly, parthenolide was able to increase the OCR
and reserve capacity in PZ cells, whereas parthenolide had no effect on PC3 OCR. Finally, the
cytotoxicity of parthenolide was tested in all the cell lines using an MTT assay, which requires
active mitochondria. As shown in Fig. 2F, parthenolide was toxic to all the cancer cells but not to
the normal cell lines. Taken together, these results suggest that changes in cellular redox status
and mitochondrial function may be a cause for the differential biological effects of parthenolide
on cancer and normal cells.
Keap1 is susceptible to parthenolide-mediated redox modification. Keap1, a redox sensitive
protein, has been reported to play an important role in cell survival under oxidative stress (29).
To investigate whether parthenolide modifies Keap1 function, a Keap1 antibody linked to biotin
was used to immunoprecipitate redox-modified Keap1 protein, and the presence of oxidized (-S-
S-) and reduced (-SH) cysteine residues was detected using a secondary antibody linked to
streptavidin. In the three normal cell lines, parthenolide increased the oxidized form of Keap1
but decreased the reduced form of Keap1 (Fig. 3A). Interestingly, the results from the three
cancer cell lines appeared to be completely opposite to results observed in normal cells treated
with parthenolide: the level of the oxidized form was decreased, but the level of the reduced
form was increased (Fig. 3B). To verify that the observed increase in reduced Keap1 also
occurred in vivo, mouse xenograft tumor tissues with and without DMAPT treatment were also
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used for determination of Keap1 redox status. Consistent with data obtained from cultured tumor
cells treated with parthenolide, the in vivo results show that parthenolide decreased the oxidized
form of Keap1 but increased the reduced form of Keap1 in the tumors (Fig. 3C). Changes in
antioxidant proteins in mouse xenograft tumor tissues treated with DMAPT are also consistent
with the result obtained from in vitro studies (Fig. 3D), indicating that parthenolide decreases the
level of mitochondrial antioxidant proteins in prostate tumors.
Oxidization of Keap1 leads to activation of the Nrf2 pro-survival pathway in normal cells.
Activation of the Nrf2 signaling pathway through dissociation with Keap1 resulting in Nrf2
nuclear translocation is considered to be a primary pro-survival pathway in response to oxidative
stress (30, 37). To examine whether parthenolide changes Nrf2 nuclear translocation, the levels
of Nrf2 in nuclei were measured. As shown in Fig. 4A, the nuclear levels of Nrf2 were increased
in the three normal cell lines treated with parthenolide, but no changes were observed in the three
cancer cell lines. To examine whether activation of the Nrf2 pathway is a major mechanism by
which parthenolide protects normal cells against radiation injury, Keap1 and Nrf2 were silenced
in PZ cells by transfecting their siRNA (Fig. 4B, left panel). Cell survival decreased when Nrf2
was silent. IR significantly reduced cell survival but the cell survival was restored when Keap1
was silenced (Fig. 4B, right panel). These results suggest that oxidation of Keap1 and subsequent
activation of Nrf2 by parthenolide are essential for normal cell survival after radiation treatment.
Thioredoxin is necessary for parthenolide-mediated reduction of Keap1 in cancer cells. TrX
is highly expressed in cancer cells and stimulates cell growth. We previously reported that
parthenolide decreases the reduced form of TrX but increases the oxidized form of TrX in
prostate cancer cells (25). In the present study, we verify that TrX was expressed at a high level
in all three cancer cell lines whereas a low level was observed in the three non-cancer cell lines
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(Fig. 5A). Immunoprecipitation of Keap1 protein from PC3 cell extracts using a TrX antibody
suggests an interaction between Keap1 and TrX that is increased by parthenolide (Fig. 5B). To
detect whether the parthenolide-influenced reduction of Keap1 in cancer cells is dependent on
TrX, we selectively silenced TrX by transfecting its siRNA prior to parthenolide treatment (Fig.
5C, left panel). As expected, the reduced form of Keap1 was decreased, but the oxidized form of
Keap1 was increased when TrX was silent (Fig. 5C, middle and right panels). The results suggest
that TrX is interacting with Keap1 to keep Keap1 in a reduced state in parthenolide-treated cells.
To further confirm that the function of Keap1 leads to cell death in parthenolide-treated cancer
cells, a Keap1 expression construct was transfected into PC3 cells, followed by parthenolide and
IR treatments. Overexpression of Keap1 resulted in increases in cell death in both treated and
untreated cells (Fig. 5D, top panel). The levels of mitochondrial phosphoglycerate mutase 5
(PGAM5), a protein serine/threonine phosphatase that interacts with Bcl-xL in the mitochondrial
membrane (38), and Bcl-xLwere clearly decreased in the Keap1 transfected cells, but no changes
were observed in Nrf2, Ikkα and IkBα (Fig. 5D, bottom panel). These results suggest that the
parthenolide-increased reduced form of Keap1facilitates Keap1-mediated ubiquitin/proteasome-
dependent degradation of PGAM5 and Bcl-xL, which is an established mechanism for
parthenolide-mediated cell death in cancer cells.
Keap1 triggers PGAM5-mediated Bcl-xL ubiquitin degradation in parthenolide-treated
cancer cells. To further investigate the mechanism by which parthenolide enhances the
radiosensitivity of prostate cancer cells, we determined the interactions between Keap1, PGAM5
and Bcl-xL. The results demonstrate that a reduced form of Keap1, which is increased in
parthenolide-treated PC3 cells, enhanced interaction between Keap1 and PGAM5, as detected by
immunoprecipitation using a PGAM5 antibody (Fig. 6A). Bcl-xL, a prosurvival mitochondrial
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protein, was also increased in the pulled down complex (Fig. 6A). Interestingly, the proteins that
are associated with Keap1 were decreased in whole cell extracts (Fig. 6B). A time course of
parthenolide treatment shows that PGAM5 and Bcl-xL proteins were slightly increased at 12 h
but decreased at 24 and 48 h after treatment (Fig. 6C). Proteins in different cellular fractions
were also quantified (Fig. 6D). The mitochondria-associated proteins PGAM5 and Bcl-xL were
reduced by the parthenolide treatment, but no change was observed in Hsp75, a control for
mitochondrial protein. Parthenolide had no major effect on the levels of Nrf2 and Ikkα in treated
cells. These results suggest that parthenolide enhances Keap1-mediated ubiquitin/proteasome-
dependent degradation of PGAM5 and Bcl-xL (39). In addition, parthenolide increased the level
of mitochondria-associated autophagic protein LC3B, suggesting that parthenolide may enhance
the radiation sensitivity of prostate cancer cells partially through triggering the autophagy
pathway.
Because Keap1 interacts with PGAM5/Bcl-xL/Nrf2, we decided to determine the effect
of PGAM5/Bcl-xL/Nrf2 in mediating parthenolide’s effect on cancer cells. PGAM5, Bcl-xL and
Nrf2 were silenced using their siRNAs, followed by parthenolide treatment (Fig. 6E, bottom
panel). The cell survival fraction was decreased when PGAM5 or Bcl-xL was silent, which is
similar to the effect of parthenolide (Fig. 6E, top panel). No significant additive effects were
observed when parthenolide was combined with PGAM5 or Bcl-xL siRNA. In contrast, a
significant effect was observed when Nrf2 was silent. These results suggest that Keap1-mediated
PGAM5/Bcl-xL degradation, but not Nrf2 degradation, is important for parthenolide-induced
cancer cell death.
To further determine whether the function of Bcl-xL plays a major role in protecting
cancer cells against parthenolide-induced cell death, a plasmid carrying Bcl-xL cDNA was
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transfected into PC3 cells followed by parthenolide treatment (Fig. 6F, bottom panel). The
results show that expression of Bcl-xL efficiently protects cells from cytotoxicity caused by
parthenolide (Fig. 6F, top panel). Taken together, these results suggest that parthenolide
enhances the radiosensitivity of prostate cancer cells, in part, by triggering ubiquitin/proteasome-
based degradation of Bcl-xL.
In summary, parthenolide provides radiosensitization in prostate cancer cells but
radioprotection in normal cells, and the observed differential effects are mediated, in part, by
redox modification of Keap1, i.e., reducing Keap1 in cancer cells but oxidizing Keap1 in normal
cells. The distinct redox modification of Keap1 initiates different signaling pathways that affect
mitochondrial function, leading to cell survival or cell injury in response to radiation, as
illustrated in Fig. 7.
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Discussion
The majority of anticancer therapies fail because cancers develop phenotypes that are
treatment resistant and because treatments cause unwanted and/or detrimental side effects to
normal cells or to untargeted tissues. While conventional adjuvant therapies improve tumor
response to radiation therapy, they generally cause additional damage to normal tissues. Thus,
the focus of the present study is to identify adjuvant therapeutics that can reduce the side effects
of radiation therapy. Our study provides a proof-of-concept for improving the efficacy of
radiation therapy while protecting against injury to normal tissues. It has been demonstrated that
parthenolide, the anti-inflammatory phytochemical, is able to suppress tumor growth in many
organs (22-25). In addition, parthenolide appears to synergically enhance chemotherapeutic
efficiency when it is combined with taxol or cisplatin to treat lung and gastric cancer cells (23,
40). Parthenolide also sensitizes radioresistant osteosarcoma cells to radiotherapy (41). Here, we
demonstrate that DMAPT, a parthenolide prodrug, sensitized prostate cancer cells to
radiotherapy in vivo and protected normal prostate and bladder against radiation-induced tissue
injury. These results extend our previous survival studies in prostate cancer cell lines and normal
prostate epithelial cells.
ROS, as products of cell metabolism, play a dual role in tumorigenesis and tumor
suppression. The “two-faced” character of ROS has emerged as a potential source for
discovering anticancer drugs. Redox homeostasis is frequently deregulated in cancers as it is
constantly exposed to high levels of ROS compared to normal counterparts. Our data
demonstrate that constitutively elevated levels of oxidative stress in cancer cells represent a
specific vulnerability that can be selectively targeted by direct- or indirect-acting prooxidants and
antioxidants or redox modulators. Theoretically, the differential redox status of cancer cells
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18
compared to normal cells should provide a therapeutic window for selective redox intervention
via additional increases in ROS. In this context, normal and cancer cells should respond
differently to the same level of prooxidant action generated either by direct production of
oxidative species or by modulation of specific cellular targets involved in redox regulation. In
this study we demonstrate that parthenolide serves as a prooxidant and displays a selective redox
modification capability that differentially modulates cellular redox signals and targets. The
Michael acceptor reacts with a thiol group of target proteins through covalent adduction (21).
Parthenolide contains electrophilic α-methylene-γ-lactone, a bisfunctional Michael acceptor, and
displays a potential for bifunctional target alkylation and crosslinking. The present study
demonstrates the inverse effects of parthenolide on redox modification in cancer cells compared
to normal cells. Remarkably, observations of the cytotoxic and cytoprotective effects of
parthenolide are consistent with its action in the modulation of ROS levels in both cancer and
normal cells. Alteration of cellular ROS by parthenolide is attributed to functionally up- or
down-regulating antioxidant enzymes in mitochondria, which consequently regulates
mitochondrial respiration. Parthenolide is able to selectively reduce the activity of several
enzymes involved in oxidative stress removal in cancer cells, which in turn can cause ROS levels
to rise above the threshold for cell death. This finding predicts that antioxidant proteins and
mitochondria are feasible therapeutic targets.
It has been reported that parthenolide is a potent inhibitor of NF-κB, which is a ROS-
responsive transcriptional factor involved in both tumor progression and tumor resistance to
treatment through upregulation of anti-apoptotic genes, such as Bcl-2, Bcl-xL, survivin, and
XIAP (42). We previously demonstrated that NADPH oxidase-mediated inactivation of the Foxo
3 signaling pathway is involved in the parthenolide-enhanced radiosensitivity of prostate cancer
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(25). However, previous studies did not explain how parthenolide exerts such an opposing effect
in tumor and normal cells. The present study identifies Keap1 as a redox signaling sensor that
plays a pivotal role in the differential regulation of the downstream signaling targets in response
to radiation-mediated cytotoxicity in prostate cancer and normal cells. Keap1, an adaptor protein
for ubiquitin-based processing by the CUL3/RBX1-dependent E3 ubiquitin ligase complex,
functions as a sensor for thiol-reactive redox modification (43). The present study demonstrates
that stabilization of Nrf2 by oxidation of Keap1 serves as a major mechanism by which
parthenolide protects normal tissues against radiotoxicity through up-regulation of antioxidant
enzymes in mitochondria. However, Nrf2 transcriptional activation did not play a major role in
parthenolide-treated prostate cancer cells. Thus, it is interesting to note that unlike traditional
chemotherapeutic agents, parthenolide is unable to enhance resistance of prostate cancer to
radiation treatment by stimulating Nrf2 target genes.
In addition to regulating the Nrf2 signaling pathway, Keap1 is able to bind other proteins
such as p62 and PGAM5 (44). Interaction between Keap1 and p62 facilitates release of Nrf2
from the complex, which is considered to be a noncanonical cysteine-independent mechanism
for the autophagy deficiency-activated Nrf2 pathway (45). The N-terminus of PGAM5 interacts
with the Kelch domain of Keap1 and its C-terminus binds to Bcl-xL. Keap1-dependent
ubiquitination results in proteasome-dependent degradation of PGAM5 and Bcl-xL (38). Bcl-xL,
an important member of the Bcl-2 family, is a potent antiapoptotic factor that plays a crucial role
in cell survival by maintaining the electrochemical and osmotic homeostasis of mitochondria
(46). The present study demonstrates that parthenolide increases the level of reduced Keap1 and
consequently induces Keap1-dependent degradation of PGAM5 and Bcl-xL in cancer cells,
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suggesting that formation of the Keap1-PGAM5-Bcl-xL complex is a mechanism underlying the
effect of parthenolide on radiosensitization of prostate cancer cells.
Although a high rate of aerobic glycolysis in tumors, known as the Warburg effect, has
been observed in various types of cancer, cancers have functional mitochondria, and
mitochondrial respiration is necessary for cancer cell proliferation (47). Cancer cells depend on a
hyperactive metabolism to fuel their rapid growth and also on antioxidative enzymes to quench
potentially toxic ROS generated by such a high metabolic demand (48). Our results demonstrate
that parthenolide not only suppresses MnSOD and GpX, two major antioxidant enzymes in
mitochondria, but also activates Bcl-xL degradation in cancer cells, which suggests that
mitochondria are a feasible target for anticancer treatment. The present study also shows that
parthenolide may maintain normal cell survival through induction of MnSOD and GpX activity.
Thus, a more efficient and safe therapy may involve modification of cellular redox signaling by
alteration of the antioxidant response coupled to selective degradation of prosurvival members of
the Bcl2 family in cancer cells, because conventional anticancer therapy mainly causes cell
growth arrest or cell death by raising cellular ROS, which oxidizes and damages DNA, proteins
and lipids. Optimizing prototype redox chemotherapeutics from natural sources provides an
exciting opportunity to further develop even better candidates to enhance therapeutic efficacy
with less off-target toxicity.
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Acknowledgments
This work was initiated by a previous graduate student, Yulan Sun, and was supported by
National Institutes of Health grants CA49797, CA115801, and CA143428 to Daret K. St. Clair
and William St. Clair. Additional support was provided by the Edward P. Evans Foundation and
by the resources and facilities of the William S. Middleton Veterans Administration Hospital
(Madison, WI).
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Figure Legends Figure 1. The effect of parthenolide on radiosensitivities of prostate cancer and normal
cells. (A) Prostate cancer PC3 cells were injected into the flanks of nude male mice. The
resulting tumors were treated with DMAPT and IR. Tumor volume was measured and tumor
growth was calculated. (B) Time needed for tumor growth to reach 2000 mm3 volume after
treatment was calculated and plotted. (C) Mice without cancer were treated with radiation alone
(5 x 3 Gy) and DMAPT (10 mg/kg) with radiation. Prostate and bladder tissues were removed
for pathological analysis using EM. Asterisks indicate normal mitochondria and arrows indicate
mitochondria with myelin figures. M, normal mitochondria; Ly, lysosome; and V, mitochondria
with vacuoles. (D) Quantification of mitochondrial damage in mice prostate and bladder tissues.
Figure 2. The effect of parthenolide on redox homeostasis in prostate cancer and normal
cells. (A-B) Cells were treated with parthenolide and then labeled with DHE or DCF. The mean
fluorescence intensity of DHE (A) and the ratio of H2DCFDA to DCFDA (B) were determined
using flow cytometry. Concentrations of cellular superoxide and total ROS were estimated by
quantification of fluorescence intensity. Antimycin and PMA were used as positive controls for
generation of ROS. PEG-SOD was used as a control to remove superoxide generated by DHE.
PN, parthenolide. (C) The levels of antioxidant proteins were quantified by western blots. (D)
The activities of the corresponding enzymes were measured. (E) Quantification of the basal
oxygen consumption, ATP-linked oxygen consumption, the maximal OCR after the addition of
FCCP, and the reserve capacity of the cells. NS, not significant. (F) Three cancer cell lines and
three non-cancer cell lines were treated with parthenolide (PN) at the indicated concentrations.
Cell survival fraction was determined by MTT assay.
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Figure 3. Keap1 redox modification by parthenolide. (A-B) Three normal cell lines (A) and
three prostate cancer cell lines (B) were treated with saline or parthenolide (PN). Keap1 was
immunoprecipitated with its antibody and SH- and S-S moieties of Keap1 were demonstrated by
ECL detection. (C) Tumor tissues were homogenized and incubated with MPB. Reduced (SH-)
and oxidized (S-S) forms of Keap1 in the tissues were detected. (D) Tumor tissues were
homogenized and antioxidant proteins were quantified by western blots. The right panel shows
representative blots and the left panel shows the average of multiple blots.
Figure 4. Activation of Keap1-Nrf2 pathway by parthenolide in normal cells. (A)
Parthenolide (PN) increases nuclear levels of Nrf2 in normal cells but not in cancer cells.
Nuclear proteins extracted from the parthenolide-treated cell lines were immunoblotted to
quantify nuclear levels of Nrf2 using PCNA as loading control. (B) The effect of Keap1 and
Nrf2 in normal cells. PZ cells were transfected with siRNAs to knock down Keap1 or Nrf2,
respectively. After treatment with parthenolide (PN) and IR, the cell survival fraction was
quantified using Trypan blue exclusion assay (right), and the knocked-down Keap1 and Nrf2
were confirmed by western blots (left).
Figure 5. TrX-dependent Keap1 reduction by parthenolide in prostate cancer cells. (A) The
levels of TrX in normal and cancer cells before and after treatment with parthenolide (PN) were
detected by western blots. (B) After the indicated treatment, Keap1 was immunoprecipitated
using a TrX antibody. (C) PC3 cells were transfected with TrX siRNA prior to the indicated
treatment (top). SH- and S-S bands in Keap1 protein were detected as described in Fig. 3
(bottom). (D) A Keap1 cDNA construct was transfected into PC3 cells. The levels of related
proteins were detected by western blots (bottom). The effect of Keap1 on radiosensitivity was
analyzed using a Trypan blue exclusion assay (top).
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Figure 6. Degradation of PGAM5-Bcl-xL caused by parthenolide-mediated reduction of
Keap1 in prostate cancer cells. (A) PC3 cells were treated with parthenolide (PN). Keap1 and
Bcl-xL were immunoprecipitated using a PGAM5 antibody. (B) The total levels of Nrf2,
PGAM5, and Bcl-xL were quantified by western blots. (C) The levels of mitochondria-
associated proteins after parthenolide treatments. (D) Proteins in various cellular fractions were
identified with antibody specific for each protein. (E) PC3 cells were transfected with siRNA to
knock down PGAM5, Bcl-xL and Nrf2, respectively (bottom). Cell survival fraction was
quantified by Trypan blue exclusion assay (top). (F) A Bcl-xL expression construct was
transfected into PC3 cells and the expression of Bcl-xL was monitored by western blots
(bottom). Cell survival fraction was quantified by Trypan blue exclusion assay (top).
Figure 7. A proposed mechanistic model for parthenolide-mediated inverse therapeutic
effects on radiosensitivity of prostate cancer and radioresistance of normal cells.
Parthenolide sensitizes cancer cells to radiation, in part, by maintaining Keap1 in a reduced state
and enhancing its interaction with PGAM5 and Bcl-xL, resulting in degradation of Bcl-xL in
mitochondria. In contrast, parthenolide protects normal cells against radiation via oxidation of
Keap1 and release of the Nrf2 transcription factor for activation of mitochondrial antioxidant
enzymes.
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Figure 1
A CA5 x 3 Gy DMAPT + 5 x 3 Gy
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Figure 2
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Figure 3
APrECPZ-HPV-7 RWPE-1 -SH -S-S-
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Figure 4
A
PN (5 μM) - + - + - + - + - + - +
PC3 DU-145 LNCaP PZ PrEC RWPE-1
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urvi
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Figure 5
PN (5 μM) - + +
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Figure 6
A IP: PGAM5
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Figure 7
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Published OnlineFirst May 14, 2013.Cancer Res Yong Xu, Fang Fang, Sumitra Miriyala, et al. Cancer CellsOpposing Radiosensitive Effects of Parthenolide in Normal and KEAP1 Is a Redox Sensitive Target That Arbitrates the
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